1. Field of the Invention
The present invention relates to the general field of protein synthesis, and more particularly, to compositions of tRNA synthetases, analogs and derivatives, thereof, and modified tRNAs that deliver non-cognate amino acids and methods for their creation, isolation and use.
2. Description of Related Art
The scientific research community has been trying to develop techniques that will allow the synthesis of custom designed proteins. Custom designed proteins are a target due to their potential applications for use in medicinal, therapeutic, diagnostic, biotechnology, engineering, and spectroscopy. Current technology relies primarily on chemical synthesis. The invention described herein was developed as a method and composition for the enzymatic and ribosome-based synthesis of custom proteins that will overcome the shortfalls of the current background art by using tRNA synthetases, their analogs and derivatives, thereof, to aminoacylate noncognate amino acids to transfer RNAs (tRNAs, their analogs and derivatives, thereof) for incorporation of the noncognate amino acids into proteins.
The present invention includes, but is not limited to, leucyl-tRNA synthetase wild-type and mutant proteins and certain editing mutants of isoleucyl-tRNA synthetases and valyl-tRNA synthetases that can misaminoacylate their cognate tRNA molecules with noncognate amino acids. It also includes, but is not limited to mutants of leucyl-tRNA synthetase that alter specificity of the activation and aminoacylation of leucine and/or noncognate amino acids. These mutant leucyl-tRNA synthetases are those that increase specificity of leucine and/or those that increase specificity of non-leucine amino acids, increase specificity of leucine while decreasing specificity of non-leucine amino acids, and increase specificity of non-leucine amino acids, while decreasing specificity of leucine.
Presently, non-enzymatic chemical acylation methods are used that covalently link non-standard amino acids to suppressor tRNAs which interact selectively with the amber (UAG) stop codon. These tRNAs and genes containing the amber mutation were incorporated into in vitro translation systems to generate proteins with site-specific incorporation of non-standard amino acids. This non-enzymatic, chemistry-based process has been commercialized by at least one company (Cruachem, Aston, Pa.), but is laborious, costly, and limited to low yields as compared to an enzymatic based approach.
Another approach is the use of an “orthogonal” tRNA, which is not recognized by existing Escherichia coli (E. coli) aminoacyl-tRNA synthetase (aaRS). The orthogonal tRNA was created based on mutations of tRNAGln. A complementary mutant glutaminyl-tRNA synthetase (GlnRS) was evolved in vitro to specifically aminoacylate the orthogonal tRNA with glutamine. This tRNA synthetase was further proposed for re-engineering of the amino acid binding pocket to activate non-standard amino acids. A mutant tRNA synthetase that aminoacylates non-glutamine amino acids to the orthogonal tRNA would create a twenty-first aaRS-tRNA pair that could be used in vivo for large scale production of proteins that contain site-specifically inserted non-standard amino acids.
A unique tRNA synthetase/tRNA pair has been developed based on the tyrosine system. The mutant tyrosyl-tRNA synthetase (TyrRS) aminoacylates or covalently links non-standard amino acids including O-methyl-L-tyrosine to tRNAs that contain an amber suppressor anticodon. The genes for this tRNA synthetase and tRNA have been used to transform E. coli. In vivo expression of these two genes within E. coli yield intracellularly synthesized proteins, expressed from genes with amber codons that contain site-specific incorporation of O-methyl-L-tyrosine. This system was also used with aspartic acid tRNA synthetase/tRNAAsp pairs.
Another method has been to evolve ribozymes made of RNA that can aminoacylate or covalently attach amino acids to tRNAs. The ribozymes exhibiting aminoacylation activity were isolated by in vitro selection techniques (also called SELEX: systematic evolution of ligands by exponential enrichment. These ribozymes have been used to attach standard amino acids including glutamine and phenylalanine to tRNA molecules. Standard amino acids that contain modifications such as biotin groups can also be aminoacylated by the selected ribozymes. RNA molecules could be further developed to specifically aminoacylate tRNA molecules with diverse non-standard amino acids and incorporated into in vitro translation systems for protein synthesis. It is possible that they could also be adapted for aminoacylation of non-standard amino acids in vivo.
Some aaRSs have difficulty completely discriminating structurally similar amino acids that compete with the cognate amino acid. It is known that non-standard amino acids can be incorporated into proteins if they are highly similar to a standard amino acid and misactivated for aminoacylation by a native tRNA synthetase. For example, trifluoroleucine is aminoacylated to tRNALeu by E. coli leucyl-tRNA synthetase (LeuRS), wherein media was supplemented with trifluoroleucine in the absence of leucine to support bacterial cell growth. Under these growth conditions, leucine substitutions by trifluoroleucine for total protein reached levels of 92%. In another example, LeuRS mischarges norvaline in vivo and isoleucine and methionine in vitro. Leucyl-tRNA synthetase has evolved a second hydrolytic active site to edit misactivated and mischarged amino acids to ensure that the correct amino acid is aminoacylated to the cognate tRNA.
Non-cognate amino acids that are misactivated or mischarged to tRNA may also be hydrolytically edited by tRNA synthetases that are related to LeuRS. These include isoleucyl-tRNA synthetase (IleRS), valyl-tRNA synthetase (ValRS), a mutant ValRS lacking editing activity, and Archae-based LeuRS. The mutant ValRS aminoacylates cysteine, threonine, and aminobutyrate to tRNAVal. In vivo studies showed that when E. coli containing the mutant ValRS was grown on media supplemented with aminobutyrate that 24% of all of the valines within proteins were replaced by aminobutyrate.
It is possible to inactivate the editing mechanism of these tRNA synthetases to stably aminoacylate noncognate amino acids to tRNAs. Aspartic acids in the hydrolytic editing active site of LeuRS, ValRS, and IleRS were proposed to play essential roles in the editing mechanism in E. coli LeuRS. An aspartic acid that is universally conserved between IleRS, ValRS, and LeuRS was proposed to be involved in a salt bridge with the α-NH4+ on the amino acid backbone of the amino acid editing substrate based on structural modeling of ValRS. A second nearby highly conserved aspartic acid that was predicted to be essential to the pre-transfer editing mechanism is in position to form a hydrogen bond with the 2′ hydroxyl of the ribose ring based on structural information. A series of mutations of the universally conserved aspartic acid in E. coli IleRS altered or abolished its amino acid editing activity.
Problems Presented by Background Art. Chemical-based synthesis of aminoacylated tRNAs is laborious, costly, and limited to low yields. In contrast, enzymatic-based aminoacylation can be more efficient, economical, and can allow synthesis of high yields of aminoacylated tRNAs. Moreover, enzymes with altered specificity for amino acid substrates could be employed to incorporate non-standard amino acids into proteins using in vivo as well as in vitro methodologies. However, site specific incorporation of these non-standard amino acids aminoacylated to tRNAs in either in vitro or in vivo translation processes would require that the tRNA anticodon is altered to recognize a non-coding codon such as a stop codon.
Enzymatic aminoacylation of non-standard amino acids requires alteration of enzyme specificity to bind and activate noncognate amino acids. Many tRNA synthetases, including GlnRS, AspRS, ValRS, IleRS and TyrRS, require protein-anticodon interactions between the tRNA synthetase and tRNA to facilitate aminoacylation activity. Long distance coupling of amino acid binding/identity/activation and anticodon-protein interactions is thus hindered when the tRNA anticodon is changed, for example to an amber suppressor, to facilitate site specific incorporation of non-standard amino acids.
Limitations in the background art are overcome in the present invention by using LeuRS. LeuRS is one of the few tRNA synthetases that lack any dependence on specific interactions with the tRNA anticodon for substrate recognition and enzyme activity. Thus, the tRNALeu anticodon can be readily changed to interact with other non leucine-encoding codons, such as stop codons, that have been previously exploited to incorporate alternate amino acids into specific sites within a protein.
High-level in vivo expression of recombinant proteins can be compromised by infidelity of certain tRNA synthetases. For example, overexpression of recombinant hemoglobin in Escherichia coli results in the substitution of norvaline amino acid intermediates for the standard amino acid leucine. Altering leucyl-tRNA synthetase to enhance specificity of leucine and decrease specificity of norvaline would lead to a higher fidelity of site-specific leucine incorporation during recombinant protein synthesis.